1. Field of Invention
This invention relates to the method of measuring peroxidase activity.
2. Description of the Prior Art
"Peroxidase" (POD) is a general term for enzyme which catalyzes the oxidizing reaction as expressed in the following general equation. This enzyme is widely distributed in animal and vegetable tissues. EQU AH.sub.2 +H.sub.2 O.sub.2 (or CH.sub.3 OOH).fwdarw.A+2H.sub.2 O(or 2CH.sub.2 OH)
Conventionally, to measure POD activity, o-phenylenediamine (OPD) is reacted with hydrogen peroxide (H.sub.2 O.sub.2), by using POD sample. This is a color reaction which produces 2,2'-diamino-azobenzene as shown in the next equation. Consequently, from the absorbance increase at maximum absorption wavelength (.lambda. max.) of the 2,2'-diamino-azobenzene, the POD catalytic intensity, that is, the POD activity can be measured. ##STR1## To perform the above method, the reagent containing OPD and H.sub.2 O.sub.2 is used in the above enzyme reaction. This reagent is treated with a buffer liquid to have its pH value adjusted to 4.0 to 6.0, which is optimal for the enzyme reaction.
The above-mentioned reagent is prepared beforehand and kept in cold-storage in a dark place, and is then added to the sample containing POD at the actual measurement.
However, OPD and H.sub.2 O.sub.2 will react slightly without POD, to give out 2,2'-diamino-azobenzene. Furthermore, this non-enzyme reaction also can somewhat easily take place at the optimum pH level for the enzyme reaction by the POD. For this reason, the conventional reagent adjusted to POD optimum pH level will gradually react during storage. Thus, when this reagent is used, the foregoing absorbance of the reagent increases to a certain extent. For example, in the case of the conventional reagent adjusted to a pH value of 5.5 and stored at 4.degree. C., its absorbance at maximum absorbance wavelength (.lambda. max.=440 nm) of the 2,2'-diamino-azobenzene doubles in about 100 minutes as shown in FIG. 2.
As has been described above, the absorbance of the conventional reagent changes with two during storage before measurement. Thus, the POD activity cannot be measured with high accuracy.